MRNA localization and local translation are required for processes ranging from mating type switching in yeast to learning and memory in mammals, yet the mechanisms driving mRNA transport and silencing in higher eukaryotic systems remain poorly understood. Axis specification in Drosophila melanogaster provides particularly dramatic examples of coordinated mRNA transport and translational silencing. Bicoid and oskar mRNA are synthesized in a cluster of nurse cells, assemble into transport particles, and move through cytoplasmic bridges (ring canals) to the oocyte. Within the oocyte, microtubules are required for bicoid mRNA localization to the anterior pole and oskar mRNA accumulation at the posterior pole. oskar mRNA is silent until mid-oogenesis, when it is localized to the posterior pole of the oocyte. Bicoid mRNA remains translationally silent until early embryogenesis. We have developed an in vivo assay for bicoid mRNA localization that reveals multiple steps in the anterior mRNA localization pathway, and an in vitro -mRNA transport particle assembly system that recapitulates RNA behavior in vivo. These assays will be used to define RNA sequences and trans-acting proteins that drive anterior localization of bicoid mRNA (Aims 1 and 2). We have also identified a new axis specification gene, armitage, that is essential for oskar mRNA translational silencing and axis specification. The armitage gene is required for RNAi and efficient assembly of the RNA induced silencing complex (RISC), and mutations in 3 additional RNAi components also disrupt oskar mRNA silencing and axis specification. The RNAi machinery thus appears to be essential to embryonic axis specification. The function of armitage in RNAi and RISC assembly, and the role of RNAi in axis specification, are examined in Aims 3 and 4. Aim 5 addresses a possible role for ATR/Chk2 tumor suppressor pathway in regulating RNAi. These studies will thus elucidate molecular mechanisms of mRNA localization and translational silencing that are essential to embryonic axis specification.